The present invention provides methods for preparing mycolactones by fermentation and novel mycolactone compounds useful in the treatment of disease conditions. The invention relates to the fields of chemistry, molecular biology, animal and human health sciences, and medicine.
Mycobacterium ulcerans causes a severe skin disease, Buruli ulcer, which is characterized by extensive necrosis in the absence of an acute inflammatory response. The causative agent of the disease was first identified as a diffusible toxic agent (see Read et al., June 1974, Cytotoxic activity of Mycobacterium ulcerans, Infection and Immun. 9(6): 1114-1122, incorporated herein by reference). Further analysis identified that the agent is a polyketide (see George et al., Feb. 5, 1999, Mycolactone: A polyketide toxin from Mycobacterium ulcerans required for virulence, Science 283: 854-857, incorporated herein by reference). Recently, the complete structural determination of two structurally related agents, called mycolactones A and B, has been reported (see Gunawardana et al., 1999, Characterization of novel macrolide toxins, mycolactones A and B, from a human pathogen, Mycobacterium ulcerans, JACS 121: 6092-6093, incorporated herein by reference).
The cytotoxic nature of the mycolactones, as well as their ability to suppress the immune response, indicates that the compounds have therapeutic potential as anti-cancer agents and immunosuppressants (see the references cited supra and Pimsler et al., March 1988, Immunosuppressive properties of the soluble toxin from Mycobacterium ulcerans, J. Infect. Dis. 157(3): 577-580, incorporated herein by reference). However, despite extensive efforts to develop fermentation conditions suitable for large-scale growth of Mycobacterium ulcerans and related organisms such as M. bovis (see e.g. Mve-Obiang et al., 1999, Growth and cytotoxic activity by Mycobacterium ulcerans in protein-free media, FEMS Microbiol. Lett. 181: 153-157; Nyabenda et al., 1988, The production of mycobacterial antigens by homogeneous culture in a fermentor, J. Biol. Standard. 16: 259-267; Palomino and Portaels, February 1998, Effects of decontamination methods and culture conditions on viability of Mycobacterium ulcerans in the BACTEC system, J. Clin. Microbiol. 36(2): 402-408; and Palomino et al., November 1998, Effect of oxygen on growth of Mycobacterium ulcerans in the BACTEC system, J. Clin. Microbiol 36(11): 3420-3422, each of which is incorporated herein by reference), cultivation of the organism remains difficult, limiting the availability of the compounds for testing and clinical trials.
There remains a need for fermentation processes by which large scale cultures of Mycobacterium ulcerans can be obtained and from which useful quantities of mycolactones could be prepared. The present invention meets that need and provides as well, as one important benefit, novel naturally produced mycolactones in purified form that were heretofore undiscovered due to the lack of suitable fermentation processes.
In a first embodiment, the present invention provides methods for the production of mycolactones by fermentation. The methods are readily scalable and can be used to produce the mycolactones in amounts sufficient for clinical trials and commercialization. The methods enable the cultivation of Mycobacterium ulcerans in a dispersed suspension culture (e.g., fermentation or spinner flasks) and the production of the mycolactones at concentrations greater than that observed in T flasks.
In a second embodiment, the present invention provides improved media formulations that allow scale-up of cultures for the production of mycolactones and improve the production of mycolactone from Mycobacterium ulcerans. 
In a third embodiment, the present invention also provides a robust and scalable method for the purification of mycolactones based on extraction and chromatography.
In a fourth embodiment the present invention provides novel mycolactone compounds isolatable from Mycobacterium ulcerans in purified form.
These and other embodiments, modes, and aspects of the invention are described in more detail in the following brief description of the figure, detailed description of the invention, the examples, and claims set forth below.